Thomas L. Feldbush

The enzyme-linked immunosorbent assay (ELISA): a measure of antibody concentration or affinity.

Abstract The immune response of rats to 2,4-dinitrophenyl (DNP) following both primary and secondary immunization with DNP-BGG has been studied using five techniques for the quantitative measurement of antibody: the standard ELISA, amplified ELISA, Farr assay, precipitation and a double-antibody precipitin assay. In addition, antibody affinity was measured by a modification of the Farr assay. Results are consistent with the view that while the Farr assay is a good indicator of total antibody. as measured by double-antibody precipitation, both ELISA assays correlate better with changes in antibody affinity. The amplified ELISA is comparatively less correlated with antibody affinity than the standard ELISA and only the amplified ELISA is capable of detecting the low affinity antibody detectable by double-antibody precipitation and by the Farr assay. Because of the influence of affinity on measurements made by the ELISA. results are best expressed as ELISA units rather than μg antihody/ml.

Antigen modulation of the immune response: The decline of immunological memory in the absence of continuing antigenic stimulation

Abstract Irradiated rats reconstituted with immune spleen cells demonstrated a decline in their ability to mount a secondary response when challenged at various times after transfer. This decline was biphasic in nature, suggesting the existence of at least two memory cell populations: one with a half-life of 4.5 days and the other with a half-life of 40 days. When antigen (DNP-BGG) was given immediately after reconstitution and the rats rechallenged 1 mo later, a significant potentiation of immunological memory occurred. This effect of antigen appeared to be independent of its ability to stimulate the memory cells into becoming antibody-forming cells. This suggested that antigen may have at least two distinct effects on the memory cell population: (1) it can stimulate the cells to become antibody forming cells or (2) it can somehow act on the cells to potentiate their functional survival.

Complement fragments suppress lymphocyte immune responses

There has been controversy as to whether complement augments immune responses or inhibits them. In this article John Weiler and his colleagues discuss recent evidence that complement fragments can inhibit immune responses that depend upon cellular proliferation.

Antigen modulation of the immune response. VI. Rate of large memory cell appearance in lymph nodes and thoracic duct lymph.

Abstract We have studied the rate of appearance of memory B-cell subpopulations in the antigen draining lymph nodes and thoracic duct lymph of rats using 1 g velocity sedimentation and adoptive transfer. Five days after immunization 100% of the memory response was attributable to large cells. By Days 7, 14, 28, and 77 after priming the large cells contribution to the memory population dropped to 86, 35, 15, and 10% respectively. At the same time the small cell contribution rose from 20% on Day 14 to 46% on Days 28 and 77. The same results were obtained with thoracic duct lymphocytes with the large cells contributing 53% of the response on Day 7 and 20% on Day 150. Appropriate controls were included to show that differential suppression was not responsible for these results. Furthermore, when purified large memory cells were passaged through intermediate hosts for 7 to 11 days, between 76 and 81% of the large cells matured into medium or small lymphocytes. These data show that the earliest memory cells formed after antigen encounter are the blast-like large lymphocytes and that these evolve, through a series of antigen-independent events, first into medium and then small lymphocytes. A model of memory cell development incorporating these results and the results of others is presented.

Antigen modulation of the immune response. IV. Selective triggering of antibody production and memory cell proliferation.

Abstract When memory cells are transferred to syngeneic irradiated recipients and then challenged at various times after transfer, a precipitous decline in the ability of these cells to mount a secondary response is seen. Using this model we have investigated some of the influences which antigen can exert on the memory cell population. The results indicate that antigen may: 1) either stimulate the memory cells to proliferate and form new memory cells or stimulate memory cells to become antibody forming cells and 2) selectively trigger the memory cells for low or high affinity antibody production. This selective antigen triggering appeared to depend upon its concentration: high dose antigen challenge led to the production of large amounts of lower affinity antibody but stimulated less memory cell proliferation while low dose challenge showed just the opposite. Control experiments indicated that recruitment of new memory cells from a virgin precursor population was not responsible for these observations. Our results thus suggest that an asymmetrical division of memory cells is occurring in which antigen can exert selective influences in much the same way as seen with virgin precursor cells.

Antigen modulation of the immune response. III. Evaluation of the hypothetical short-lived memory cell.

Abstract The putative short-lived memory cells, whose existence has been suggested by the results of secondary adoptive transfer experiments, was investigated. On the basis of the following evidences we have concluded that the short-lived memory cell is probably an artifact of the adoptive transfer technique: (a) when immune thoracic duct lymphocytes, known to consist predominantly of long-lived memory cells, were transferred to irradiated recipients and challenged at various times after transfer, approximately 80–90% of the initial response was absent by Day 14 challenge; (b) Preirradiating adoptive recipients with increasing dose of X-irradiation tended to lengthen the observed half life of memory cells; (c) single or multiple treatments of immune donors with 0.3 mg Vinblastin before transfer resulted in neither a depression of the initial secondary response nor an alteration in the rate of decline of the memory potential; (d) reconstitution of irradiated hosts with normal spleen cells one day before transfer of memory cells and challenge resulted in inhibition of the adoptive secondary response; and (e) the transfer of memory cells to antigen free intermediate hosts, in which they were allowed to reside for one day or fourteen days before transfer to irradiated recipients, resulted in only a slight decline in their capacity to respond. We propose that the rapid decline of memory potential in adoptive recipients challenged at various times after transfer is due to modulating effects by the hosts as it recovers from irradiation. These effects may be the result of cell crowding or the loss of irradiation-produced stimulatory factors. The relevance of these findings to adoptive transfer systems in general and the secondary response of intact animals is discussed.

Lymphokine regulation of surface Ia expression on rat B cells

Class II major histocompatibility antigens (Ia) play a major role in regulating T-B cell interactions; therefore, regulation of the amount of Ia on B cells may be an important point of control in the immune response. Mitogens in human and murine systems have been reported to increase Ia expression on B cells, and in the mouse the lymphokine BSF-1 (IL-4) markedly enhances Ia expression. This report describes studies of lymphokine and mitogen regulation of class II expression on rat B cells. Mitogens known to activate resting B cells, such as DxS/LPS, anti-IgM, STM, and Con A, induced increases in Ia expression. Highly purified murine IL-4 was found to have no Ia-enhancing activity on rat B cells, although the same preparation increased Ia expression eightfold on murine B cells. This confirms other recent reports that IL-4 is a species-specific lymphokine and will not cross even narrow phylogenetic barriers. Rat B cells were not refractory to lymphokine-induced enhancement of Ia expression, since lymphokine(s) contained in a Con A-induced supernatant enhanced Ia expression. Furthermore, murine IL-5-containing B151-CFS was able to markedly increase Ia expression on resting rat and mouse B cells. This activity was not lost after heat inactivation of B151-TRF2, indicating that B151-TRF1 (IL-5-like activity) was probably responsible for the increase in Ia expression. These results suggest Ia expression on rat B cells, like human and murine B cells, is an early activation event which is regulated by signals which act on resting B cells. Furthermore, while IL-4 is important in Ia regulation, it is not the only lymphokine involved, since the IL-4-free, B151-K12 supernatant was able to enhance Ia expression on resting rat and mouse B cells.

Antigen modulation of the immune response: V. Generation of large memory cells in antigen draining lymph nodes☆

Abstract We have studied the distribution of memory B cell subpopulations by using 1 g velocity sedimentation and adoptive transfer. When the non-antigen-draining mesenteric lymph nodes were examined 4 weeks after intraperitoneal immunization with DNPBGG, large memory cells were present in only very low numbers. However, when the draining parathymic nodes were removed, a significant enrichment of large memory cell activity was seen. When these results were corrected for the cell yields in each 1 g separated fraction we found that 59% of the total memory cells were small, 36% medium and 5% large in the mesenteric lymph node preparations and 40% were small, 46% medium and 14% large in the parathymic lymph node suspensions. When popliteal lymph nodes were removed after footpad immunization, 32% of the total memory cell activity was in the small cell fraction while 49% was in the medium fraction and 18% in the large cell fraction. Control experiments were also run to show that the shift in the velocity sedimentation profile of the various memory cell populations was not an artifact of the adoptive transfer system nor a result of selective antigen triggering. From these results it would appear that the size distribution of memory cells depends upon the source of cells studied, large memory cells being found predominantly only in lymph nodes draining the site of antigen injection. Since the large memory cells can also be found in the thoracic duct lymph after footpad immunization but not after intraperitoneal immunization, it is suggested that the larger cells can circulate to other lymphoid tissues but cannot recirculate.

Antigen modulation of the secondary immune response: VI. Contribution of cells recruited from precursor pool to expression of memory

Abstract The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory cells.

Radiation-induced alterations in murine lymphocyte homing patterns: I. Radiolabeling studies☆

In vitro x-irradiation of /sup 51/Cr-labeled spleen, lymph node, bone marrow, or thymus cells was found to alter their subsequent in vivo distribution significantly in syngeneic BDF/sub 1/ mice. Irradiated cells demonstrated an increased distribution to the liver and a significantly lower retention in the lungs. Cells going to the lymph nodes or Peyers patches showed a significant exposure-dependent decrease in homing following irradiation. Irradiated lymph node cells homed in greater numbers to the spleen and bone marrow, while irradiated cells from other sources showed no preferential distribution to the same tissues. Sampling host tissues at various times after irradiation and injection did not demonstrate any return to normal patterns of distribution. The alterations in lymphocyte homing observed after in vitro irradiation appear to be due to the elimination of a selective population of lymphocytes or membrane alterations of viable cells, and the detection of these homing changes is in turn dependent upon the relative numbers of various lymphoid subpopulations which are obtained from different cell sources. Radiation-induced alterations in the normal homing patterns of lymphoid cells may thus be of considerable importance in the evaluation of subsequent functional assays in recipient animals.